Compositions and methods of treating cardiac hypertrophy and heart failure

ABSTRACT

A method of treating cardiac hypertrophy and/or heart failure in a subject includes administering to the subject a therapeutically effective amount of a REV-ERBα agonist.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/457,579, filed Feb. 10, 2017, the subject matter of which isincorporated herein by reference in their entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant Nos.RO1HL119195 and KO1HL123551, awarded by The National Institutes ofHealth. The United States government has certain rights to theinvention.

BACKGROUND

Cardiac hypertrophy is an adaptive response to pressure or volumestress, mutations of sarcomeric (or other) proteins, or loss ofcontractile mass from prior infarction. Hypertrophic growth accompaniesmany forms of heart disease, including ischemic disease, hypertension,congestive heart failure, valvular disease and subsequent cardiac death.In these types of cardiac pathology, pressure overload-inducedconcentric hypertrophy is believed to have a compensatory function bydiminishing wall stress and oxygen consumption. At the same time,ventricular hypertrophy is associated with significantly increased riskof heart failure and malignant arrhythmia.

REV-ERBα, also known as NR1D1 (nuclear receptor subfamily 1, group D,member 1) is a transcriptional repressor. REV-ERBα is highly expressedin the liver, skeletal muscle, adipose tissue, and the brain, inmammals, participating in the development and circadian regulation ofthese tissues. REV-ERBα regulates gene transcription by directly bindingto target response elements (RevREs), comprises an A/T-rich flankfollowed by AGGTCA.

SUMMARY

Embodiments described herein relate to compositions and methods for usein the prevention and treatment of cardiac hypertrophy and heartfailure. It was found that pharmacological activation of REV-ERBαselectively suppresses aberrant pathologic gene expression and preventscardiomyocyte hypertrophy. In vivo, REV-ERBα activation preventsdevelopment of cardiac hypertrophy, reduces fibrosis, and haltsprogression of advanced heart failure. Accordingly, in some embodimentsa method of treating and/or preventing cardiac hypertrophy and/or heartfailure in a subject in need thereof includes administering to thesubject a therapeutically effective amount of a REV-ERBα agonist.

In some embodiments, the REV-ERBα agonist is selected from the groupconsisting of 1,1-DimethylethylN-[(4-chlorophenyl)methyl]-N-[(5-nitro-2-thienyl)methyl])glycinate(SR6452);N-Benzyl-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;N-Benzyl-N-(3,4-dichlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N,N-dimethylacetamide,SR9009, SR9011 and pharmaceutically acceptable salts thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 (A-D) illustrate an image and plots showing REV-ERB agonistprevented the phenylephrine-induced (PE-induced) cardiomyocytehypertrophy through transcriptional repression of the fetal geneprogram. (A-C) Neonatal rat ventricular myocytes (NRVMs) were pretreatedwith vehicle or SR9009 for 24 hours and then treated with PE for 48hours. (A) Immunofluorescent staining with α-actinin (green). Scale bar:30 μM. (B) Quantification of cell size. *P=0.02, n=42-114. (C) qPCR ofNppa and Nppb. *P=0.00009 (Nppa), *P=0.00007 (Nppb), n =3. (D) NRVMswere pretreated with vehicle or GSK4112 for 24 hours and then treatedwith PE for 48 hours. qPCR of Nppa and Nppb. *P=0.00001 (Nppa),*P=0.0003 (Nppb), n=3. Statistical differences were determined by2-tailed Student's t test. Data are presented as mean ±SEM. Multiplecomparison is corrected for by using Holm-Sidak method, with a =0.05.

FIGS. 2 (A-D) illustrate plots showing REV-ERB agonist amelioratecardiac dysfunction in pressure-overload models. Six-week followup of27-gauge TAC performed on 8-weekold mice, vehicle, or SR9009, which weregiven daily starting 1 day after surgery. n =5. (A) Ejection fraction(EF), *P<0.05. (B) Fraction shortening (FS), *P<0.05. (C) Left ventricleinternal diameter end diastole. (D) Left ventricle internal diameter endsystole. *P<0.05. Statistical differences were determined by 2-tailedStudent's t test. Data are presented as mean ±SEM. Multiple comparisonis corrected for by using Holm-Sidak method, with α=0.05.

FIGS. 3 (A-I) illustrate images and plots showing REV-ERB agonistameliorate cardiac hypertrophy in pressure overload models. Six-weekfollowup of 27-gauge TAC was performed on 8-weekold mice, vehicle, orSR9009, which were given daily starting one day after surgery. n=5. (A)Left ventricle mass, corrected, *P<0.05. (B) Heart weight normalized totibia length, *P<0.05. (C) Representative pictures of vehicle- andSR9009-treated hearts harvested at 6 weeks. Scale bar: 5 mm. (D) Wheatgerm agglutinin staining. Scale bar: 30 μM. (E) Quantified muscle fibersize in arbitrary units, *P<0.05. 41. (F) Fibrosis stained with GomoriTrichrome stain, representative pictures. Vehicle, left column andmiddle column. SR9009, right column. Scale bar: 400 μM. (G)Quantification of fibrotic area. (H) TUNEL staining, representativepicture. Scale bar: 20 μM. (I) Quantification of TUNEL signal persection. Statistical differences were determined by 2-tailed Student's ttest. Data are presented as mean ±SEM. Multiple comparison is correctedfor by using Holm-Sidak method, with α=0.05.

FIGS. 4 (A-B) illustrate a schematic of an experimental design of atwenty-eight—gauge TAC with 11-week followup (B) a graph showin ejectionfraction, *P=0.01. Statistical differences were determined by 2-tailedStudent's t test. Data are presented as mean ±SEM. Multiple comparisonis not corrected for. n=5.

FIG. 5 illustrates a plot showing a REV-ERB agonist prevented theendothelin-1-induced (ET-1-induced) hypertrophy in human inducedpluripotent stem cell-differentiated cardiomyocytes (iPS-CM). iPS-CMwere pretreated with vehicle or SR9009 for 24 hours and then treatedwith ET-1 for 48 hours. qPCR of Nppb. *P=0.01 (untreated), **P=0.003(ET-1), n =3. Statistical differences were determined by 2-tailedStudent's t test. Data are presented as mean ±SEM. Multiple comparisonis corrected for by using Holm-Sidak method, with α=0.05.

DETAILED DESCRIPTION OF THE INVENTION

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisapplication belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and include,without limitation, intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

“Treat”, “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a patient at risk for or afflicted with adisease, including improvement in the condition through lessening orsuppression of at least one symptom, delay in progression of thedisease, prevention or delay in the onset of the disease, etc.

The terms “prevent,” “preventing,” or “prevention” are art-recognizedand include precluding, delaying, averting, obviating, forestalling;stopping, or hindering the onset, incidence, severity, or recurrence ofa disease, disorder or condition from occurring in a subject, which maybe predisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it. Preventing a condition related to a diseaseincludes stopping the condition from occurring after the disease hasbeen diagnosed but before the condition has been diagnosed.

The term “pharmaceutical composition” refers to a formulation containingthe disclosed compounds in a form suitable for administration to asubject. In a preferred embodiment, the pharmaceutical composition is inbulk or in unit dosage form. The unit dosage form is any of a variety offorms, including, for example, a capsule, an IV bag, a tablet, a singlepump on an aerosol inhaler, or a vial. The quantity of active ingredient(e.g., a formulation of the disclosed compound or salts thereof) in aunit dose of composition is an effective amount and is varied accordingto the particular treatment involved. One skilled in the art willappreciate that it is sometimes necessary to make routine variations tothe dosage depending on the age and condition of the patient. The dosagewill also depend on the route of administration. A variety of routes arecontemplated, including oral, pulmonary, rectal, parenteral,transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal,intranasal, inhalational, and the like. Dosage forms for the topical ortransdermal administration of a compound described herein includespowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, nebulized compounds, and inhalants. In a preferred embodiment,the active compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The compounds described herein can also be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compound,which releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds can bedelivered in prodrug form. Thus, the compounds described herein areintended to cover prodrugs of the presently claimed compounds, methodsof delivering the same and compositions containing the same. “Prodrugs”are intended to include any covalently bonded carriers that release anactive parent drug in vivo when such prodrug is administered to asubject. Prodrugs are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds wherein a hydroxy, amino, sulfhydryl, carboxy, orcarbonyl group is bonded to any group that may be cleaved in vivo toform a free hydroxyl, free amino, free sulfhydryl, free carboxy or freecarbonyl group, respectively. Prodrugs can also include a precursor(forerunner) of a compound described herein that undergoes chemicalconversion by metabolic processes before becoming an active or moreactive pharmacological agent or active compound described herein.

A “patient,” “subject,” or “host” to be treated by the subject methodmay mean either a human or non-human animal, such as a mammal, a fish, abird, a reptile, or an amphibian. Thus, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal. In another aspect, the subject is aprematurely born mammal treated with prolonged supplemental oxygen. Apatient refers to a subject afflicted with a disease or disorder.

The terms “prophylactic” or “therapeutic” treatment is art-recognizedand includes administration to the host of one or more of thetherapeutic compositions described herein. If it is administered priorto clinical manifestation of the unwanted condition (e.g., disease orother unwanted state of the host animal) then the treatment isprophylactic, i.e., it protects the host against developing the unwantedcondition, whereas if it is administered after manifestation of theunwanted condition, the treatment is therapeutic (i.e., it is intendedto diminish, ameliorate, or stabilize the existing unwanted condition orside effects thereof).

The terms “therapeutic agent”, “drug”, “medicament” and “bioactivesubstance” are art-recognized and include molecules and other agentsthat are biologically, physiologically, or pharmacologically activesubstances that act locally or systemically in a patient or subject totreat a disease or condition. The terms include without limitationpharmaceutically acceptable salts thereof and prodrugs. Such agents maybe acidic, basic, or salts; they may be neutral molecules, polarmolecules, or molecular complexes capable of hydrogen bonding; they maybe prodrugs in the form of ethers, esters, amides and the like that arebiologically activated when administered into a patient or subject.

The phrase “therapeutically effective amount” or “pharmaceuticallyeffective amount” is an art-recognized term. In certain embodiments, theterm refers to an amount of a therapeutic agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, the term refers to thatamount necessary or sufficient to eliminate, reduce or maintain a targetof a particular therapeutic regimen. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation.

As used herein, the term “cardiac hypertrophy” is used in its ordinarymeaning as understood by the medical community and is often associatedwith increased risk of morbidity and mortality. It generally refers tothe process in which adult cardiac myocytes respond to stress throughhypertrophic growth. Such growth is characterized by cell size increaseswithout cell division or proliferation, assembling of additionalsarcomeres within the cell to maximize force generation, and anactivation of a fetal cardiac gene program.

As used herein, the term “heart failure” is broadly used to mean anycondition that reduces the ability of the heart to pump blood. As aresult, congestion and edema develop in the tissues. Most frequently,heart failure is caused by decreased contractility of the myocardium,resulting from reduced coronary blood flow; however, many other factorsmay result in heart failure, including damage to the heart valves,vitamin deficiency, and primary cardiac muscle disease. The terms “heartfailure,” “manifestations of heart failure,” “symptoms of heartfailure,” and the like are used broadly to encompass all of the sequelaeassociated with heart failure, such as shortness of breath, pittingedema, an enlarged tender liver, engorged neck veins, pulmonary ralesand the like including laboratory findings associated with heartfailure.

Embodiments described herein relate to compositions and methods for usein the prevention and treatment of cardiac hypertrophy, heart failure,and/or related pathologies. Pathologies related to cardiac hypertrophyand heart failure include ischemic heart disease, hypertension, cardiacfibrosis, valvular disease and additional cardiovascular diseases, suchas restrictive cardiomyopathies, valvuloseptal disorders, cardiacdilation and genetic syndromes of dysfunctional heart action.

It was found that pharmacological activation of REV-ERBα selectivelysuppresses aberrant pathologic gene expression and preventscardiomyocyte hypertrophy. In vivo, REV-ERBα activation preventsdevelopment of cardiac hypertrophy, reduces fibrosis, and haltsprogression of advanced heart failure. Accordingly, a method oftreating, preventing, reversing, ameliorating and/or delaying cardiachypertrophy, heart failure, and/or related pathologies in a subjecthaving, suspected of having, or at risk of such cardiac hypertrophy,heart failure, and/or related pathologies includes administering to thesubject in need thereof a therapeutically effective amount of a REV-ERBαagonist. In some embodiments, the REV-ERBα agonist can be administeredat an amount effective to upregulate at least one of REV-ERBαexpression, activity, and subcellular localization and/or reduce cardiachypertrophy, cardiomyocyte death, and/or cardiac fibrosis.

The REV-ERBα agonist can include any agent that either directly orindirectly activates REV-ERBα and that can facilitate REV-ERBα torecruit its corepressor NCoR and repress downstream targets. TheREV-ERBα agonist can inhibit cardiomyocyte hypertrophy and cellularstress in a cell-autonomous fashion. The REV-ERBα agonist can includecompounds (e.g., small molecules, ligands, proteins, enzymes,antibodies, nucleic acids, etc.) that increase or enhance the activityof REV-ERBα in vivo and/or in vitro. REV-ERBα agonists can also includecompounds that exert their effect on REV-ERBα activity via alteringexpression, via post-translational modifications, or by other means.Agonists of REV-ERBα can comprise molecules which, when bound toREV-ERBα, increase or prolong the activity of REV-ERBα (e.g., increasethe nuclear localization and/or nuclear activity of REV-ERBα). Agonistsof REV-ERBα according to certain embodiments can include proteins,nucleic acids, carbohydrates, small molecules, or any other moleculeswhich activate REV-ERBα.

In some embodiments, the REV-ERBα agonist can directly upregulateREV-ERBα activity or expression in cardiac cells of a subject. Incertain embodiments, the REV-ERBα agonist can include a synthetic ligand(e.g., small molecule) that upregulates REV-ERBα activity or expression.For instance, in certain embodiments, the REV-ERBα agonist can include asynthetic ligand for REV-ERBα. Modulators of REV-ERBα agonist activityand/or expression have been disclosed, e.g., in WO 2013/033310, thecontents of which are incorporated herein by reference. AdditionalREV-ERBα agonists can be identified by screening potential compounds,e.g., as described in Grant et al. (2010), ACS Chem. Biol. 5(10):925-32,the contents of which are incorporated herein by reference. Suchpotential compounds can include, for example, variants of any REV-ERBαagonist compound specifically disclosed herein.

Examples of synthetic agonists for REV-ERBα include 1,1-DimethylethylN-[(4-chlorophenyl)methyl]-N-[(5-nitro-2-thienyl)methyl])glycinate(GSK4112),N-Benzyl-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;N-Benzyl-N-(3,4-dichlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine,2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N,N-dimethylacetamide;or combinations thereof. Other examples of synthetic agonists forREV-ERBα include SR9009, SR9011, GSK2945, GSK0999, GSK5072, and/orGS2667, which are described in WO 2013/033310 and are structurallyrelated to the foregoing synthetic agonists. In specific embodiments,the REV-ERBα agonist is SR9009.

Certain embodiments utilize the administration of synthetic REV-ERBαagonist1,1-Dimethylethyl-N-[(4-chlorophenyl)methyl]-N-[(5-nitro-2-thienyl)methyl])glycinate(EC₅₀=250 nM) or salt thereof, which is known as SR6452 or GSK4112 andis commercially available from Sigma Aldrich (USA).1,1-Dimethylethyl-N-[(4-chlorophenyl)methyl]-N-[(5-nitrothienyl)methyl])glycinate will hereinafter be referred to as “SR6452”.REV-ERBα agonist SR6452 enhances recruitment of nuclear receptorco-repressor (NCoR) peptide to REV-ERBα. The structure of SR6452 is asfollows:

In certain embodiments, the REV-ERBα agonist can includeN-Benzyl-N-(4-chlorobenzyl)-l-(5-nitrothiophen-2-yl)methanamine. Thestructure ofN-Benzyl-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine is asfollows:

In certain embodiments, the REV-ERBα agonist can includeN-Benzyl-N-(3,4-dichlorobenzyl)-1 -(5-nitrothiophen-2-yl)methanamine.The structure ofN-Benzyl-N-(3,4-dichlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine isas follows:

In certain embodiments, the REV-ERBα agonist can include2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N,N-dimethylacetamide.The structure of2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N,N-dimethylacetamideis as follows:

In some embodiments, the REV-ERBα agonist is a 6-substituted[1,2,4]triazolo[4,3-b]pyridazine, such as compounds of general formula(I) described in U.S. Patent Application Publication No. 2017/0296548A1,which is incorporated by reference in its entirety

In certain embodiments, the REV-ERBα agonist is a compound related toSR6452, such as SR9009 or SR9011. See, e.g., WO 2013/033310.

In other embodiments, the REV-ERBα agonist can include a naturalmolecule (e.g., Heme modulators). For instance, a natural REV-ERBαagonist can include enzymes, antibodies, proteins, nucleic acids,carbohydrates, small molecules, or combination thereof includingupstream regulators (both known such as glycogen synthase kinase 3(GSK3) and/or novel).

To improve efficacy and/or reduce side effects, the REV-ERBα agonist canbe linked to another moiety that functions as a carrier and/or targetingmoiety. The other moiety can selectively targets cardiac cells. Thecarrier/targeting moiety can, in some embodiments, increase the serumhalf-life of the REV-ERBα agonist. In other embodiments, thecarrier/targeting moiety can inncrease the serum half-life of theREV-ERBα agonist and selectively targets cardiac cells.

As noted previously, REV-ERBα agonist according to certain embodimentscan indirectly modulate REV-ERBα activity or expression. In suchembodiments, a REV-ERBα agonist can target or interact with a componentof the cardiac cells upstream to REV-ERBα expression or a component thatregulates nuclear transport of REV-ERBα. This component, for instance,can then function to modulate REV-ERBα expression or activity.Accordingly, the REV-ERBα agonist effectively modulates REV-ERBαactivity or expression indirectly through interaction with the upstreamcomponent of the cardiac cell that subsequently affects the activity orexpression of REV-ERBα due to the initial interaction with the REV-ERBαagonist. In certain such embodiments, the REV-ERBα agonist can compriseenzymes, antibodies, proteins, nucleic acids, carbohydrates, smallmolecules, or combinations thereof. The component of the cardiac cellupstream to REV-ERBα expression can comprise, for example, an enzyme,antibody, protein, nucleic acid, carbohydrate, or combinations thereofincluding upstream regulators (known regulators, such as GSK3, and/ornovel regulators).

In some embodiments, a therapeutically effective amount of a REV-ERBαagonist can include an amount sufficient to achieve its intendedpurpose. More specifically, a therapeutically effective amount caninclude an amount effective to prevent development of cellularhypertrophy or alleviate the existing symptoms in the subject beingtreated. A therapeutically effective amount can vary based on a range offactors (e.g., route of administration, patient's age, patient's weight,severity of disorder, etc.) and determination thereof is well within thecapability of those skilled in the art.

For instance, a therapeutically effective amount of a REV-ERBα agonistcan be estimated initially from cell culture assays. For example, a dosecan be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ (the dose where 50% of thecells show the desired effects) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inmammals (e.g., humans).

A therapeutically effective amount of a REV-ERBα agonist can also referto that amount of the compound that results in amelioration of symptomsor a prolongation of survival in a patient. Toxicity and therapeuticefficacy of a REV-ERBα agonist can be determined by standardpharmaceutical procedures in cell cultures or experimental animals(e.g., for determining the LD50-the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio between LD50and ED50. Compounds, which exhibit high therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosages or amounts for usein mammals (e.g., humans). The dosage or amount of a REV-ERBα agonistpreferably lies within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage or amount mayvary within this range depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. Dosage amount and interval may beadjusted individually to provide plasma levels of the active moietywhich are sufficient to maintain the desired effects.

In cases of local administration or selective uptake, the effectivelocal concentration of the REV-ERBα agonist may not be related to plasmaconcentration.

The amount of REV-ERBα agonist -containing composition administered can,of course, be dependent upon several factors including the subject beingtreated, on the subject's weight, the severity of the affliction, themanner of administration and the judgment of the prescribing physician.

In certain embodiments, a REV-ERBα agonist can be administered to amammal having, suspected of having, or at risk of cardiac hypertrophy,heart failure, and/or related pathologies at an amount sufficient toenhance REV-ERBα agonist expression, activity, and/or subcellularlocation. In accordance with certain embodiments, REV-ERBα agonistexpression and/or activity can be enhanced or increased by at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or more. For instance, REV-ERBα agonistexpression and/or activity can be enhanced or increased from at leastany of the following: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and/or at most aboutany of the following 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (e.g., 5-100%, 10-90%,20-80%, etc.). In other embodiments, REV-ERBα agonist subcellularlocalization can be enhanced or increased (e.g., shifted to the nucleus)by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. For instance, REV-ERBαagonist subcellular localization can be enhanced or increased (e.g.,shifted to the nucleus) such that the amount of REV-ERBα in a particularlocation (e.g., the nucleus) is from at least any of the following: 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, to atmost about any of the following 80%, 85%, 90%, 95%, 100% (e.g., 30-100%,40-90%, 50-80%, etc.).

In certain embodiments, the methods can include the administration of atherapeutically effective amount of a REV-ERBα agonist, in which theamount of the REV-ERBα agonist comprises an amount effective to haltcardiac cell hypertrophy, preferably concomitantly with modulation ofREV-ERBα expression, activity, and/or subcellular location. Forinstance, the rate at which hypertrophic cardiomyocyte growth can beginto reduce until no noticeable further growth is realized. In thisregard, these embodiments can provide a means to effectively impede orstop the further progression or severity of cardiac hypertrophy andrelated pathologies.

In some embodiments, the methods can include the administration of atherapeutically effective amount of a REV-ERBα agonist, in which theamount of the REV-ERBα agonist comprises an amount sufficient to haltand/or reduce cardiac fibrosis, preferably concomitantly with anupregulation or overexpression of REV-ERBα. In such embodiments, thedegree of fibrosis can be reduced to beneficially reduce theconsequences associated with intracardiac fibrosis such as thecontractile function of the cardiac tissue.

In certain embodiments, the subject (e.g., human) being treated has beendiagnosed as having cardiac hypertrophy. In other embodiments, however,the subject (e.g., human) being treated may not technically have cardiachypertrophy but may be exhibiting symptoms similar to or associated withcardiac hypertrophy. In certain embodiments, the subject (e.g., human)being treated may be identified as being at risk of developing cardiachypertrophy, cardiac hypertrophy associated cardiovascular diseaseand/or heart failure in view of diagnosis of conditions known toultimately lead to development of cardiovascular disease. In suchembodiments, the administration of a REV-ERBα agonist n can beneficiallyfacilitate or prevent development of cardiac hypertrophy, cardiachypertrophy associated cardiovascular disease and/or heart failure.

In some embodiments, the REV-ERBα agonist can provided in apharmaceutical composition with a wide variety of pharmaceuticallyacceptable carriers or excipients. The particular carriers or excipientscan be varied depending on various factors including route ofadministration, presence or absence of a carrier/targeting moiety, anddesired delivery system (e.g., sustained release, timed-released,immediate release, selective release, etc.). For example, thecomposition can be made to suit the desired mode of administration.Pharmaceutically acceptable carriers can be determined, in part, by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulation recipes of pharmaceutical compositionscontaining one or more REV-ERBα agonists. For example, thepharmaceutical carrier may comprise a virus, a liposome (e.g., cationiclipids mixed with a REV-ERBα agonist to form liposomes carrying theREMA), or a polymer (e.g., cationic polymers such as DEAE-dextran orpolyethylenimine in which the REV-ERBα agonist complexes with thepolycation and the complex is taken up by the cell via endocytosis).

The administration of a pharmaceutical composition that includes aREV-ERBα agonist may be carried out by known methods, wherein a desiredmolecule is introduced into a desired target cell in vitro or in vivo.In general, methods of administering small molecules, nucleic acids,enzymes and proteins are well known in the art. REV-ERBα agonistcompositions in accordance with some embodiments can be administered bya number of routes including, but not limited to: oral, intravenous,intraperitoneal, intraarterial, intramuscular, transdermal,subcutaneous, topical, sublingual, or rectal means. Alternatively, theREV-ERBα agonist can be administered using a cellular vehicle, such ascells “loaded” with the REV-ERBα agonist ex vivo.

Administration of the compositions described herein may be accomplishedby any acceptable method which allows a REV-ERBα agonist to reach itstarget. Any acceptable method known to one of ordinary skill in the artmay be used to administer a composition to the subject. Theadministration may be localized (i.e., to a particular region,physiological system, tissue, organ, or cell type) or systemic,depending on the condition being treated. In certain embodiments, thetargeted tissue comprises a cardiomyocyte.

Injections can be, for example, intravenous, intradermal, subcutaneous,intramuscular, or intraperitoneal. In certain embodiments, theinjections can be given at multiple locations if desired. In certainembodiments, the compositions can be delivered by implantation.Implantation can include inserting implantable drug delivery systems,e.g., microspheres, hydrogels, polymeric reservoirs, cholesterolmatrixes, polymeric systems, e.g., matrix erosion and/or diffusionsystems and non-polymeric systems, e.g., compressed, fused, orpartially-fused pellets. In certain embodiments, the compositions can bedelivered orally or sublingually. In certain embodiments, thecompositions can be delivered by inhalation. Inhalation can includeadministering the composition with an aerosol in an inhaler, eitheralone or attached to a carrier that can be absorbed. For systemicadministration, it may be preferred that the composition is encapsulatedin liposomes.

In some embodiments, the REV-ERBα agonist delivery systems can beprovided in a manner which enables tissue-specific uptake of theREV-ERBα agonist. Techniques include using tissue or organ localizingdevices, such asstents having drug delivery capability and configured asexpansive devices or stent grafts.

In some embodimens, a nucleic acid encoding a REV-ERBα agonist can beprovided in a a vector. Such vectors can include a sequence encoding aparticular REV-ERBα agonist of choice and in vivo expression elements.Vectors can include, but are not limited to, plasmids, cosmids,phagemids, viruses, other vehicles derived from viral or bacterialsources that have been manipulated by the insertion or incorporation ofthe nucleic acid sequences for producing the desired REV-ERBα agonist,and free nucleic acid fragments which can be attached to these nucleicacid sequences. Viral and retroviral vectors are a preferred type ofvector according to certain embodiments and include, but are not limitedto, nucleic acid sequences from the following viruses: retroviruses,such as: Moloney murine leukemia virus; Murine stem cell virus, Harveymurine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus;adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses;Epstein-Barr viruses; papilloma viruses; herpes viruses; vacciniaviruses; polio viruses; and RNA viruses such as any retrovirus. One ofskill in the art can readily employ other vectors known in the art.

Viral vectors are generally based on non-cytopathic eukaryotic virusesin which nonessential genes have been replaced with the nucleic acidsequence of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Retroviruses have been approved for human gene therapy trials.Genetically altered retroviral expression vectors have general utilityfor the high-efficiency transduction of nucleic acids in vivo.

Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in Kriegler, M.,“Gene Transfer and Expression, A Laboratory Manual,” W.H. Freeman Co.,New York (1990) and Murry, E. J. Ed. “Methods in Molecular Biology,”vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In certain embodiments, a therapeutically effective amount of one ormore REV-ERBα agonists can be delivered to a mammal via ananoparticle-based drug delivery system. For instance, nanoparticlesused as carriers for REV-ERBα agonists can provide the benefits of highstability, high carrier capacity, feasibility of incorporation of bothhydrophilic and hydrophobic substances, and feasibility of variableroutes of administration, including oral application and inhalation. Incertain embodiments, the nanoparticles can also be designed to allowcontrolled (sustained) release of the REV-ERBα agonists from the matrix.The aforementioned properties of nanoparticles, according to certainembodiments of the present invention, can provide improvement ofbioavailability and/or reduction of the dosing frequency. Nanoparticlesfor the purpose of REV-ERBα agonist delivery can be defined as submicron(<1 μm) colloidal particles. The colloidal particles can includemonolithic nanoparticles (nanospheres) in which the REV-ERBα agonist isadsorbed, dissolved, or dispersed throughout the matrix and nanocapsulesin which the REV-ERBα agonist is confined to an aqueous or oily coresurrounded by a shell-like wall. Alternatively, the REV-ERBα agonistscan be covalently attached to the surface or into the matrix.Nanoparticles, according to certain embodiments of the presentinvention, can be made from biocompatible and biodegradable materialssuch as polymers, either natural (e.g., gelatin, albumin) or synthetic(e.g., polylactides, polyalkylcyanoacrylates), or solid lipids. In thebody of the mammal being treated, the REV-ERBα agonists loaded innanoparticles can be released from the matrix by a variety of mechanismsincluding, for example, diffusion, swelling, erosion, degradation, orcombinations thereof. In one embodiment, the composition comprising oneor more REV-ERBα agonists can be perfused directly through the targetedtissue. For example, the composition containing a REV-ERBα agonist canbe perfused directly through a body organ, without introducing theREV-ERBα agonist into the body's general circulation, removing them fromthe organ with effluent blood and transporting the contaminated blood toan extracorporeal circuit where the blood is treated to remove thecontamination, and returning the treated blood to the body. In someembodiments, such a process may help prevent undesirable levels of theREV-ERBα agonist from entering the body's general circulation whiledelivering effective doses to the cardiomyocytes. Methods of perfusingactive agents through a body organ, are described in greater detail inU.S. Pat. No. 5,069,662, the contents of which are incorporated byreference in their entirety.

In certain embodiments, the compositions can be delivered using abioerodible implant by way of diffusion or by degradation of a polymericmatrix. In certain embodiments, the administration of the compositionsmay be designed so as to result in sequential exposures to the REV-ERBαagonist over a certain time period, for example, hours, days, weeks,months or years. This may be accomplished, for example, by repeatedadministrations or by a sustained or controlled release delivery systemin which a REV-ERBα agonist is delivered over a prolonged period withoutrepeated administrations. Administration of the compositions using sucha delivery system may be, for example, by oral dosage forms (e.g.,tablet, capsule, etc.), bolus injections, transdermal patches orsubcutaneous implants. Maintaining a substantially constantconcentration of the REV-ERBα agonist may be preferred in some cases.

Other delivery systems include, but are not limited to, time-release,delayed release, sustained release, or controlled release deliverysystems (e.g., tablets, capsules, etc.). Such systems may avoid repeatedadministrations in many cases, increasing convenience to the subject andthe physician. Many types of release delivery systems are available andknown to those of ordinary skill in the art. They include, for example,polymer-based systems such as polylactic and/or polyglycolic acids,polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides,polyorthoesters, polyhydroxybutyric acid, and/or combinations of these.

Microcapsules of the foregoing polymers containing nucleic acids aredescribed in, for example, U.S. Pat. No. 5,075,109. Other examplesinclude nonpolymer systems that are lipid-based including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono-, di- and triglycerides; hydrogel release systems;liposome-based systems; phospholipid based-systems; silastic systems;peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; or partially fused implants.Specific examples include, but are not limited to, erosional systems inwhich a synthetic compound (e.g., SR6452, SR9009, SR9011) is containedin a formulation within a matrix (for example, as described in U.S. Pat.Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and5,239,660), or diffusional systems in which an active component controlsthe release rate (for example, as described in U.S. Pat. Nos. 3,832,253,3,854,480, 5,133,974 and 5,407,686). The compositions may be as, forexample, microspheres, hydrogels, polymeric reservoirs, cholesterolmatrices, or polymeric systems. In certain embodiments, the system mayallow sustained or controlled release of the composition to occur, forexample, through control of the diffusion or erosion/degradation rate ofthe formulation containing the REV-ERBα agonist. In addition, apump-based hardware delivery system may be used to deliver one or moreembodiments.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Use of a long-term release implant may be particularly suitable in someembodiments. “Long-term release,” as used herein, means that the implantcontaining the composition is constructed and arranged to delivertherapeutically effective levels of the composition for at least 30 or45 days, and preferably at least 60 or 90 days, or even longer in somecases. Long-term release implants are well known to those of ordinaryskill in the art, and include some of the release systems describedabove.

Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g., by means ofan appropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. The doseadministered to a patient is sufficient to effect a beneficialtherapeutic response in the patient over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose can be determined by the efficacy of the particularformulation, and the activity, stability or serum half-life of theREV-ERBα agonist employed and the condition of the patient, as well asthe body weight or surface area of the patient to be treated. The sizeof the dose can also be determined by the existence, nature, and extentof any adverse side-effects that accompany the administration of aparticular composition in a particular patient.

Optimal precision in achieving concentrations of the therapeutic regimen(e.g., a pharmaceutical composition comprising one or more REV-ERBαagonists) within the range that yields maximum efficacy with minimaltoxicity may require a regimen based on the kinetics of thepharmaceutical composition's availability to one or more target sites.Distribution, equilibrium, and elimination of a pharmaceuticalcomposition may be considered when determining the optimal concentrationfor a treatment regimen. Generally, the pharmaceutical compositions ofthe present invention may be administered in a manner that maximizesefficacy and minimizes toxicity.

Moreover, the dosage administration of the compositions of the presentinvention may be optimized using a pharmacokinetic/pharmacodynamicmodeling system. For example, one or more dosage regimens may be chosenand a pharmacokinetic/pharmacodynamic model may be used to determine thepharmacokinetic/pharmacodynamic profile of one or more dosage regimens.Next, one of the dosage regimens for administration may be selectedwhich achieves the desired pharmacokinetic/pharmacodynamic responsebased on the particular pharmacokinetic/pharmacodynamic profile. See WO00/67776, which is entirely expressly incorporated herein by reference.

More specifically, the pharmaceutical compositions may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three, or four times daily. In the case of oraladministration, the daily dosage of the compositions may be varied overa wide range from about 0.1 ng to about 1,000 mg per patient, per day.The range may more particularly be from about 0.001 ng/kg to 10 mg/kg ofbody weight per day, about 0.1-100 μg, about 1.0-50 μg or about 1.0-20mg per day for adults (at about 60 kg).

The daily dosage of the pharmaceutical compositions may be varied over awide range from about 0.1 ng to about 1000 mg per adult human per day.For oral administration, the compositions may be provided in the form oftablets containing from about 0.1 ng to about 1000 mg of the compositionor 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 milligrams ofthe composition for the symptomatic adjustment of the dosage to thepatient to be treated. An effective amount of the pharmaceuticalcomposition is ordinarily supplied at a dosage level of from about 0.1ng/kg to about 20 mg/kg of body weight per day. In one embodiment, therange is from about 0.2 ng/kg to about 10 mg/kg of body weight per day.In another embodiment, the range is from about 0.5 ng/kg to about 10mg/kg of body weight per day. The pharmaceutical compositions may beadministered on a regimen of about 1 to about 10 times per day.

In the case of injections, it is usually convenient to give by anintravenous route in an amount of about 0.01 μg-30 mg, about 0.01 μg-20mg or about 0.01-10 mg per day to adults (at about 60 kg). In the caseof other animals, the dose calculated for 60 kg may be administered aswell.

Doses of a pharmaceutical composition of the present invention canoptionally include 0.0001 μg to 1,000 mg/kg/administration, or 0.001m to100.0 mg/kg/administration, from 0.01m to 10 mg/kg/administration, from0.1m to 10 mg/kg/administration, including, but not limited to, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or100-500 mg/kg/administration or any range, value or fraction thereof, orto achieve a serum concentration of 0.1 , 0.5, 0.9, 1.0, 1.1, 1.2, 1.5,1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0,6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9,11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0,5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14,14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9,19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration or any range, value or fraction thereof.

As a non-limiting example, treatment of humans or animals can beprovided as a onetime or periodic dosage of a composition of the presentinvention 0.1 ng to 100 mg/kg such as 0.0001, 0.001, 0.01, 0.1 0.5, 0.9,1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70,80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, oralternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, or 52, or alternatively or additionally,at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 years, or any combination thereof, using single,infusion or repeated doses.

Specifically, the pharmaceutical compositions may be administered atleast once a week over the course of several weeks. In one embodiment,the pharmaceutical compositions are administered at least once a weekover several weeks to several months. In another embodiment, thepharmaceutical compositions are administered once a week over four toeight weeks. In yet another embodiment, the pharmaceutical compositionsare administered once a week over four weeks.

More specifically, the pharmaceutical compositions may be administeredat least once a day for about 2 days, at least once a day for about 3days, at least once a day for about 4 days, at least once a day forabout 5 days, at least once a day for about 6 days, at least once a dayfor about 7 days, at least once a day for about 8 days, at least once aday for about 9 days, at least once a day for about 10 days, at leastonce a day for about 11 days, at least once a day for about 12 days, atleast once a day for about 13 days, at least once a day for about 14days, at least once a day for about 15 days, at least once a day forabout 16 days, at least once a day for about 17 days, at least once aday for about 18 days, at least once a day for about 19 days, at leastonce a day for about 20 days, at least once a day for about 21 days, atleast once a day for about 22 days, at least once a day for about 23days, at least once a day for about 24 days, at least once a day forabout 25 days, at least once a day for about 26 days, at least once aday for about 27 days, at least once a day for about 28 days, at leastonce a day for about 29 days, at least once a day for about 30 days, orat least once a day for about 31 days.

Alternatively, the pharmaceutical compositions may be administered aboutonce every day, about once every 2 days, about once every 3 days, aboutonce every 4 days, about once every 5 days, about once every 6 days,about once every 7 days, about once every 8 days, about once every 9days, about once every 10 days, about once every 11 days, about onceevery 12 days, about once every 13 days, about once every 14 days, aboutonce every 15 days, about once every 16 days, about once every 17 days,about once every 18 days, about once every 19 days, about once every 20days, about once every 21 days, about once every 22 days, about onceevery 23 days, about once every 24 days, about once every 25 days, aboutonce every 26 days, about once every 27 days, about once every 28 days,about once every 29 days, about once every 30 days, or about once every31 days. The pharmaceutical compositions of the present invention mayalternatively be administered about once every week, about once every 2weeks, about once every 3 weeks, about once every 4 weeks, about onceevery 5 weeks, about once every 6 weeks, about once every 7 weeks, aboutonce every 8 weeks, about once every 9 weeks, about once every 10 weeks,about once every 11 weeks, about once every 12 weeks, about once every13 weeks, about once every 14 weeks, about once every 15 weeks, aboutonce every 16 weeks, about once every 17 weeks, about once every 18weeks, about once every 19 weeks, about once every 20 weeks.

Alternatively, the pharmaceutical compositions may be administered aboutonce every month, about once every 2 months, about once every 3 months,about once every 4 months, about once every 5 months, about once every 6months, about once every 7 months, about once every 8 months, about onceevery 9 months, about once every 10 months, about once every 11 months,or about once every 12 months.

Alternatively, the pharmaceutical compositions may be administered atleast once a week for about 2 weeks, at least once a week for about 3weeks, at least once a week for about 4 weeks, at least once a week forabout 5 weeks, at least once a week for about 6 weeks, at least once aweek for about 7 weeks, at least once a week for about 8 weeks, at leastonce a week for about 9 weeks, at least once a week for about 10 weeks,at least once a week for about 11 weeks, at least once a week for about12 weeks, at least once a week for about 13 weeks, at least once a weekfor about 14 weeks, at least once a week for about 15 weeks, at leastonce a week for about 16 weeks, at least once a week for about 17 weeks,at least once a week for about 18 weeks, at least once a week for about19 weeks, or at least once a week for about 20 weeks.

Alternatively the pharmaceutical compositions may be administered atleast once a week for about 1 month, at least once a week for about 2months, at least once a week for about 3 months, at least once a weekfor about 4 months, at least once a week for about 5 months, at leastonce a week for about 6 months, at least once a week for about 7 months,at least once a week for about 8 months, at least once a week for about9 months, at least once a week for about 10 months, at least once a weekfor about 11 months, or at least once a week for about 12 months.

Therapeutic compositions that include one or more REV-ERBα agonists canoptionally be tested in one or more appropriate in vitro and/or in vivoanimal models of disease, to confirm efficacy, tissue metabolism, and toestimate dosages, according to methods well known in the art. Inparticular, dosages can be initially determined by activity, stabilityor other suitable measures of treatment vs. non-treatment (e.g.,comparison of treated vs. untreated cells or animal models), in arelevant assay. Formulations are administered at a rate determined bythe LD50 of the relevant formulation, and/or observation of anyside-effects of the REV-ERBα agonist at various concentrations, e.g., asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

In some embodiments, the pharmaceutical compositions (e.g., the REV-ERBαagonist) can be combined with one or more therapeutic agents. Inparticular, the compositions described herein and other therapeuticagents can be administered simultaneously or sequentially by the same ordifferent routes of administration. The determination of the identityand amount of therapeutic agent(s) for use in the methods describedherein can be readily made by ordinarily skilled medical practitionersusing standard techniques known in the art. In specific embodiments, aREV-ERBα agonist of the present invention can be administered incombination with an effective amount of a therapeutic agent that treatscardiac hypertrophy and/or any heart disease associated with cardiachypertrophy.

Therapeutic agents include, but are not limited to, beta blockers,anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators,hormone antagonists, inotropes, diuretics, endothelin antagonists,calcium channel blockers, phosphodiesterase inhibitors, ACE inhibitors,angiotensin type 2 antagonists and cytokine blockers/inhibitors, andHDAC inhibitors.

More specifically, a REV-ERBα agonist may be combined with anothertherapeutic agent including, but not limited to, anantihyperlipoproteinemic agent, an antiarteriosclerotic agent, anantithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmicagent, an antihypertensive agent, a vasopressor, a treatment agent forcongestive heart failure, an antianginal agent, an antibacterial agentor a combination thereof.

In specific embodiments, a REV-ERBα agonist may be combined with anantihyperlipoproteinemic agent including aryloxyalkanoic/fibric acidderivative, a resin/bile acid sequesterant, a HMG CoA reductaseinhibitor, a nicotinic acid derivative, a thyroid hormone or thyroidhormone analog, a miscellaneous agent or a combination thereof, acifran,azacosterol, benfluorex, β-benzalbutyramide, carnitine, chondroitinsulfate, clomestrone, detaxtran, dextran sulfate sodium, eritadenine,furazabol, meglutol, melinamide, mytatrienediol, ornithine, γ-oryzanol,pantethine, pentaerythritol tetraacetate, phenylbutyramide, pirozadil,probucol (lorelco), β-sitosterol, sultosilic acid-piperazine salt,tiadenol, triparanol and xenbucin.

A REV-ERBα agonist may be combined with an antiarteriosclerotic agentsuch as pyridinol carbamate. In other embodiments, a REV-ERBα agonistmay be combined with an antithrombotic/fibrinolytic agent including, butnot limited to anticoagulants (acenocoumarol, ancrod, anisindione,bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfatesodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidenedicoumarol, fluindione, heparin, hirudin, lyapolate sodium, oxazidione,pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide,tioclomarol and warfarin); anticoagulant antagonists, antiplateletagents (aspirin, a dextran, dipyridamole (persantin), heparin,sulfinpyranone (anturane) and ticlopidine (ticlid)); thrombolytic agents(tissue plaminogen activator (activase), plasmin, pro-urokinase,urokinase (abbokinase) streptokinase (streptase), anistreplase/APSAC(eminase)); thrombolytic agent antagonists or combinations thereof).

In other embodiments, a REV-ERBα agonist may be combined with a bloodcoagulant including, but not limited to, thrombolytic agent antagonists(amiocaproic acid (amicar) and tranexamic acid (amstat); antithrombotics(anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin,fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride,tedelparin, ticlopidine and triflusal); and anticoagulant antagonists(protamine and vitamine K1).

Alternatively, a REV-ERBα may be combined with an antiarrhythmic agentincluding, but not limited to, Class I antiarrythmic agents (sodiumchannel blockers), Class II antiarrythmic agents (beta-adrenergicblockers), Class II antiarrythmic agents (repolarization prolongingdrugs), Class IV antiarrhythmic agents (calcium channel blockers) andmiscellaneous antiarrythmic agents. Non-limiting examples of sodiumchannel blockers include Class IA (disppyramide (norpace), procainamide(pronestyl) and quinidine (quinidex)); Class IB (lidocaine (xylocalne),tocamide (tonocard) and mexiletine (mexitil)); and Class ICantiarrhythmic agents, (encamide (enkaid) and fiecamide (tambocor)).

Non-limiting examples of a beta blocker (also known as a β-adrenergicblocker, a β-adrenergic antagonist or a Class II antiarrhythmic agent)include acebutolol (sectral), alprenolol, amosulalol, arotinolol,atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol,bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrinehydrochloride, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol(brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propanolol (inderal), sotalol (betapace), sulfmalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In certain aspects, thebeta blocker comprises an aryloxypropanolamine derivative. Non-limitingexamples of aryloxypropanolamine derivatives include acebutolol,alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol,bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol,metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol,pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol.Non-limiting examples of an agent that prolongs repolarization, alsoknown as a Class III antiarrhythmic agent, include amiodarone(cordarone) and sotalol (betapace).

Non-limiting examples of a calcium channel blocker, otherwise known as aClass IV antiarrythmic agent, include an arylalkylamine (e.g.,bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline,verapamil), a dihydropyridine derivative (felodipine, isradipine,nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) apiperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) ora micellaneous calcium channel blocker such as bencyclane, etafenone,magnesium, mibefradil or perhexyline. In certain embodiments a calciumchannel blocker comprises a long-acting dihydropyridine(nifedipine-type) calcium antagonist.

Non-limiting examples of miscellaneous antiarrhymic agents includeadenosine (adenocard), digoxin (lanoxin), acecamide, ajmaline,amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine,capobenic acid, cifenline, disopyranide, hydroquinidine, indecamide,ipatropium bromide, lidocaine, lorajmine, lorcamide, meobentine,moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidinepolygalacturonate, quinidine sulfate and viquidil.

In other embodiments, a REV-ERBα agonist may be combined with anantihypertensive agent including, but not limited to, alpha/betablockers (labetalol (normodyne, trandate)), alpha blockers,anti-angiotensin II agents, sympatholytics, beta blockers, calciumchannel blockers, vasodilators and miscellaneous antihypertensives.

Non-limiting examples of an alpha blocker, also known as an a-adrenergicblocker or an a-adrenergic antagonist, include amosulalol, arotinolol,dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin,labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin andyohimbine. In certain embodiments, an alpha blocker may comprise aquinazoline derivative. Non-limiting examples of quinazoline derivativesinclude alfuzosin, bunazosin, doxazosin, prazosin, terazosin andtrimazosin.

Non-limiting examples of anti-angiotension II agents include angiotensinconverting enzyme inhibitors and angiotension II receptor antagonists.Non-limiting examples of angiotensin converting enzyme inhibitors (ACEinhibitors) include alacepril, enalapril (vasotec), captopril,cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril,perindopril, quinapril and ramipril. Non-limiting examples of anangiotensin II receptor blocker, also known as an angiotension IIreceptor antagonist, an ANG receptor blocker or an ANG-II type-1receptor blocker (ARBS), include angiocandesartan, eprosartan,irbesartan, losartan and valsartan. Non-limiting examples of asympatholytic include a centrally acting sympatholytic or a peripherallyacting sympatholytic. Non-limiting examples of a centrally actingsympatholytic, also known as a central nervous system (CNS)sympatholytic, include clonidine (catapres), guanabenz (wytensin)guanfacine (tenex) and methyldopa (aldomet). Non-limiting examples of aperipherally acting sympatholytic include a ganglion blocking agent, anadrenergic neuron blocking agent, a β-adrenergic blocking agent or anα1-adrenergic blocking agent. Non-limiting examples of a ganglionblocking agent include mecamylamine (inversine) and trimethaphan(arfonad). Non-limiting of an adrenergic neuron blocking agent includeguanethidine (ismelin) and reserpine (serpasil). Non-limiting examplesof a β-adrenergic blocker include acenitolol (sectral), atenolol(tenormin), betaxolol (kerlone), carteolol (cartrol), labetalol(normodyne, trandate), metoprolol (lopressor), nadanol (corgard),penbutolol (levatol), pindolol (visken), propranolol (inderal) andtimolol (blocadren). Non-limiting examples of alphal-adrenergic blockerinclude prazosin (minipress), doxazocin (cardura) and terazosin(hytrin).

In certain embodiments, an antihypertensive agent may comprise avasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or aperipheral vasodilator). In particular embodiments, a vasodilatorcomprises a coronary vasodilator including, but not limited to,amotriphene, bendazol, benfurodil hemisuccinate, benziodarone,chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole,droprenilamine, efloxate, erythrityl tetranitrane, etafenone, fendiline,floredil, ganglefene, herestrol bis(P-diethylaminoethyl ether),hexobendine, itramin tosylate, khellin, lidoflanine, mannitolhexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate,pentrinitrol, perhexyline, pimethylline, trapidil, tricromyl,trimetazidine, trolnitrate phosphate and visnadine.

In certain aspects, a vasodilator may comprise a chronic therapyvasodilator or a hypertensive emergency vasodilator. Non-limitingexamples of a chronic therapy vasodilator include hydralazine(apresoline) and minoxidil (loniten). Non-limiting examples of ahypertensive emergency vasodilator include nitroprusside (nipride),diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten)and verapamil.

Non-limiting examples of miscellaneous antihypertensives includeajmaline, γ-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, acryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate,mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone,muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, aprotoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodiumnitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase andurapidil. In certain aspects, an antihypertensive may comprise anarylethanolamine derivative (amosulalol, bufuralol, dilevalol,labetalol, pronethalol, sotalol and sulfmalol); a benzothiadiazinederivative (althizide, bendroflumethiazide, benzthiazide,benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone,cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide,fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide,meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazideand trichlonnethiazide); a N-carboxyalkyl(peptide/lactam) derivative(alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat,fosinopril, lisinopril, moveltipril, perindopril, quinapril andramipril); a dihydropyridine derivative (amlodipine, felodipine,isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine andnitrendipine); a guanidine derivative (bethanidine, debrisoquin,guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine,guanochlor, guanoxabenz and guanoxan); a hydrazines/phthalazine(budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine,hydralazine, pheniprazine, pildralazine and todralazine); an imidazolederivative (clonidine, lofexidine, phentolamine, tiamenidine andtolonidine); a quanternary ammonium compound (azamethonium bromide,chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate),pentamethonium bromide, pentolinium tartrate, phenactropinium chlorideand trimethidinium methosulfate); a reserpine derivative (bietaserpine,deserpidine, rescinnamine, reserpine and syrosingopine); or asulfonamide derivative (ambuside, clopamide, faro semide, indapamide,quinethazone, tripamide and xipamide).

In other embodiments, a REV-ERBα agonist may be combined with avasopressor. Vasopressors generally are used to increase blood pressureduring shock, which may occur during a surgical procedure. Non-limitingexamples of a vasopressor, also known as an antihypotensive includeamezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine,etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine,pholedrine and synephrine.

A REV-ERBα agonist may be combined with treatment agents for congestiveheart failure including, but not limited to, anti-angiotension IIagents, afterload-preload reduction treatment (hydralazine (apresoline)and isosorbide dinitrate (isordil, sorbitrate)), diuretics, andinotropic agents.

Non-limiting examples of a diuretic include a thiazide orbenzothiadiazine derivative (e.g., althiazide, bendroflumethazide,beizthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide,ethiazide, ethiazide, fenquizone, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, meticrane, metolazone,paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide),an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide,mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurouschloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines(e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom,protheobromine, theobromine), steroids including aldosterone antagonists(e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative(e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone,diphenylmethane-4,4′-disulfonamide, disulfamide, ethoxzolamide,furosemide, indapamide, mefruside, methazolamide, piretanide,quinethazone, torasemide, tripamide, xipamide), a uracil (e.g.,aminometradine, amisometradine), a potassium sparing antagonist (e.g.,amiloride, triamterene) or a miscellaneous diuretic such as aminozine,arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine,isosorbide, mannitol, metochalcone, muzolimine, perhexyline, ticrnafenand urea.

Non-limiting examples of a positive inotropic agent, also known as acardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline,aminone, benfurodil hemisuccinate, bucladesine, cerberosine,camphotamide, convallatoxin, cymarin, denopamine, deslanoside,digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine,dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin,glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside,metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine,prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin,strphanthin, sulmazole, theobromine and xamoterol.

In particular aspects, an intropic agent is a cardiac glycoside, abeta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limitingexamples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin(crystodigin). Non-limiting examples of a β-adrenergic agonist includealbuterol, bambuterol, bitolterol, carbuterol, clenbuterol,clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex),dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Non-limiting examples of a phosphodiesterase inhibitorinclude aminone (inocor).

In certain aspects, the secondary therapeutic agent may comprise asurgery of some type, which includes, for example, preventative,diagnostic or staging, curative and palliative surgery. Surgery, and inparticular a curative surgery, may be used in conjunction with othertherapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for hypertrophy, vascular andcardiovascular diseases and disorders are well known to those of skillin the art, and may comprise, but are not limited to, performing surgeryon an organism, providing a cardiovascular mechanical prostheses,angioplasty, coronary artery reperfusion, catheter ablation, providingan implantable cardioverter defibrillator to the subject, mechanicalcirculatory support or a combination thereof. Non-limiting examples of amechanical circulatory support that may be used in the present inventioncomprise an intra-aortic balloon counterpulsation, left ventricularassist device or combination thereof.

Alternatively, therapeutic agents that can be administered incombination therapy with one or more REV-ERBα agonists include, but arenot limited to, anti-inflammatory, anti-viral, anti-fungal,anti-mycobacterial, antibiotic, amoebicidal, trichomonocidal, analgesic,anti-neoplastic, anti-hypertensives, anti-microbial and/or steroiddrugs, to treat cardiac hypertrophy and/or any heart disease associatedwith cardiac hypertrophy. In some embodiments, patients are treated witha REV-ERBα agonist in combination with one or more of the following;β-lactam antibiotics, tetracyclines, chloramphenicol, neomycin,gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid,cortisone, hydrocortisone, betamethasone, dexamethasone, fluocortolone,prednisolone, triamcinolone, indomethacin, sulindac, acyclovir,amantadine, rimantadine, recombinant soluble CD4 (rsCD4), anti-receptorantibodies (e.g., for rhinoviruses), nevirapine, cidofovir (Vistide™),trisodium phosphonoformate (Foscarnet™), famcyclovir, pencyclovir,valacyclovir, nucleic acid/replication inhibitors, interferon,zidovudine (AZT, Retrovir™), didanosine (dideoxyinosine, ddl, Videx™),stavudine (d4T, Zerit™), zalcitabine (dideoxycytosine, ddC, Hivid™),nevirapine (Viramune™), lamivudine (Epivir™, 3TC), pro tease inhibitors,saquinavir (Invirase™, Fortovase™), ritonavir (Norvir™), nelfmavir(Viracept™), efavirenz (Sustiva™), abacavir (Ziagent™), amprenavir(Agenerase™) indinavir (Crixivan™), ganciclovir, AzDU, delavirdine(Kescriptor™), kaletra, trizivir, rifampin, clathiromycin,erythropoietin, colony stimulating factors (G-CSF and GM-CSF),non-nucleoside reverse transcriptase inhibitors, nucleoside inhibitors,adriamycin, fluorouracil, methotrexate, asparagyinase and combinationsforegoing.

In another aspect, the REV-ERBα agonists may be combined with othertherapeutic agents including, but not limited to, immunomodulatoryagents, anti-inflammatory agents (e.g., adrenocorticoids,corticosteroids (e.g., beclomethasone, budesonide, flunisolide,fluticasone, triamcinolone, methlyprednisolone, prednisolone,prednisone, hydrocortisone), glucocorticoids, steroids, non-steriodalantiinflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and COX-2inhibitors), and leukotreine antagonists (e.g., montelukast, methylxanthines, zafirlukast, and zileuton), β2-agonists (e.g., albuterol,biterol, fenoterol, isoetharie, metaproterenol, pirbuterol, salbutamol,terbutalin formoterol, salmeterol, and salbutamol terbutaline),anticholinergic agents (e.g., ipratropium bromide and oxitropiumbromide), sulphasalazine, penicillamine, dapsone, antihistamines,anti-malarial agents (e.g., hydroxychloroquine), other anti-viralagents, and antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, erythomycin, penicillin, mithramycin, and anthramycin (AMC)).

In various embodiments, a REV-ERBα agonist in combination with a secondtherapeutic agent may be administered less than 5 minutes apart, lessthan 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hoursapart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hoursto 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hoursapart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96hours to 120 hours part. In particular embodiments, two or moretherapies are administered within the same patent visit.

In certain embodiments, a REV-ERBα agonist and one or more othertherapies are cyclically administered. Cycling therapy involves theadministration of a first therapy (e.g., a REV-ERBα agonist) for aperiod of time, followed by the administration of a second therapy(e.g., a second REV-ERBα agonist or another therapeutic agent) for aperiod of time, optionally, followed by the administration of a thirdtherapy for a period of time and so forth, and repeating this sequentialadministration, e.g., the cycle, in order to reduce the development ofresistance to one of the therapies, to avoid or reduce the side effectsof one of the therapies, and/or to improve the efficacy of thetherapies. In certain embodiments, the administration of the combinationtherapy of the present invention may be repeated and the administrationsmay be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6months.

The dosages of a pharmaceutical composition disclosed herein may beadjusted when combined to achieve desired effects. On the other hand,dosages of the pharmaceutical composition and various therapeutic agentsmay be independently optimized and combined to achieve a synergisticresult wherein the pathology is reduced more than it would be if eitherwere used alone.

The following example is included to demonstrate different embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the example, which follow representtechniques discovered by the inventors to function well in the practiceof the claimed embodiments, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the claims.

EXAMPLE

We set out to determine whether REV-ERBα functions in hypertrophicgrowth of cardiomyocytes that accompanies many forms of heart diseaseand whether a REV-ERBα modulation is effective in the prevention ofcardiomyocyte cellular hypertrophy.

Animals

WT C57BL/6J mice were purchased from the Jackson Laboratory at the ageof 7 weeks and allowed to acclimate in the Case Western ReserveUniversity for 2 weeks prior to the experiments described below.Sprague-Dawley rat pups were purchased from the Charles RiverLaboratories at 2 days of age and sacrificed for NRVMs isolation uponarrival. Preparation and administration of SR9009 and GSK41112

SR9009 was synthesized and purified in the laboratory of Thomas Burris(Department of Pharmacology and Physiology, St. Louis University, St.Louis, Mo., USA). For in vitro experiments, SR9009 was dissolved in DMSOand administered at 5 M; GSK4112 (EMD Millipore) was dissolved in DMSOand administered at 10 M using an equal volume of DMSO as control. Forin vivo experiments, SR9009 was dissolved in 5% DMSO/10% Cremaphor EL(Sigma-Aldrich, C5135)/85% PBS in a working solution at 10 mg/ml.Micewere injected at a dose of 100 mg/kg/day given i.p. once daily atzeitgeber time 8 (ZT8). The diluent without SR9009 of the same volumewas used as control.

TAC

All mice were C57BL/6J littermate males aged 9 weeks at the start of theexperiment. Mice were anesthetized with ketamine/xylazine, mechanicallyventilated (Harvard apparatus), and subjected to thoracotomy. The aorticarch was constricted between the left and right carotid arteries using a7.0 silk suture and a 27- or 28-guage needle as previously described(20). Chemicals were obtained from Sigma-Aldrich.

Echocardiography

For transthoracic echocardiography, mice were anesthetized with 1%inhalational isofluorane and imaged using the Vevo 770 High ResolutionImaging System (Visual Sonics Inc.) and the RMV-707B 30 MHz probe.Measurements were obtained from M-mode sampling, and integratedEKVimages were taken in the LV short axis at the mid-papillary level.

Blood Pressure

Conscious tail-vein systolic blood pressure was measured using theBP2000 Blood Pressure Analysis System (Visitech Systems Inc.) asrecommended by the manufacturer. To allow mice to adapt to theapparatus, we performed daily blood pressure measurements for 1 weekprior to beginning experiments.

NRVMs and CF Culture

NRVMs and CFs were isolated from the hearts of 2-day-old Sprague-Dawleyrat pups (Charles River Laboratories). The cells were differentiallypreplated for 1.5 hr. The nonattached cells were collected as NRVM andreplated with 48 hr exposure to BrdU to suppress nonmyocytes. Theattached cells were passaged 2-3 times to obtain CFs. NRVMs wereinitially plated in growth medium (DMEM supplemented with 10% FBS, 100U/ml penicillin-streptomycin, and 2 mM L-glutamine) for 48 hr andmaintained in serum-free media thereafter (DMEM supplemented with 1%insulin-transferrin-selenium liquid media supplement [Sigma-Aldrich,13146], 100 U/ml penicillin-streptomycin). For hypertrophic stimulation,NRVM were incubated with SR9009 or GSK4112 versus DMSO for 24 hr,followed by stimulation with PE (100 μM) for the indicated time. CFswere cultured in DMEM supplemented with 10% FBS, 100 U/mlpenicillin-streptomycin, and 2 mM L-glutamine and stimulated with TGFβ1(eBioscience).

Human Cardiomyocytes Culture

We purchased human induced pluripotent stem cell-differentiatedcardiomyocytes from Cellular Dynamics (iCell). We followedmanufacturer's protocol and measured NPPB expression 24 hr after ET-1(10 nM, Sigma-Aldrich) induction.

Cell Area Measurements

NRVM were plated on glass coverslips in 6-well dishes at a density of1×105 cells/ml. After treatments, cells were fixed in ice cold methanol,permeabilized with PBST/0.3% Triton X-100, and blocked in PBST/5% horseserum. Primary antibody was anti-α-actinin (Sigma-Aldrich, A7811) at1:500. Secondary antibody (anti-mouse Alexa 488, Life Technologies) wasused at 1:1,000 dilution. Coverslips were mounted on glass slides withmounting media containing DAPI. Quantitation of cardiomyocyte cellsurface area was performed on anti-actinin-stained cardiomyocytes usingfluorescent microscopy and ImagePro software. Analysis consisted of atleast 200 cardiomyocytes in 20-30 fields at 400×magnification. Threeindependent experiments were performed.

Histological Analysis

Short-axis heart sections from the midventricle were fixed in PBS/4%paraformaldehyde and embedded in paraffin. Fibrosis was visualized usingGomori's Trichrome staining kit (Sigma-Aldrich) with quantification offibrotic area using ImagePro software. TUNEL staining was performedusing the CardioTACS In Situ Apoptosis Detection kit (R&D Systems)according to manufacturer's instructions. Cryopreserved sections wereused for myocardial capillary staining, which was performed in frozen LVsections using anti-PECAM-1 antibodies (1:200, BD Pharmingen, MEC13.3).The cardiomyocyte cross-sectional area was determined by staining withWGA Alexa 594 (Life Technologies) and analyzed using ImagePro software.

RNA Purification and Quantitative PCR

For tissue RNA, a 10-20 mg piece of mouse heart at apex was preserved inRNA Later stabilization reagent (Qiagen) followed by mechanicaldisruption/homogenization in Trizol (Life Technologies) on a TissueLyser(Qiagen) using stainless steel beads (Qiagen). RNA was purified from theaqueous phase using the miRNAeasy kit (Qiagen) following manufacturer'sinstructions. For cellular samples, total RNA from NRVM was isolatedusing the High Pure RNA isolation kit (Roche Diagnostics) with on-columnDNAase treatment according to manufacturer's directions. Purified RNAwas reverse transcribed to complementary DNA using the iScript™ RTSupermix (Bio-Rad) following manufacturer's protocol. qPCR was performedusing TaqMan chemistry (Taqman Fast Advanced Master Mix, AppliedBiosystems) and labeled probes from the Roche Universal Probe LibrarySystem on Applied Biosystems ViiA 7. Relative expression was calculatedusing the ΔΔCt-method with normalization to Ppib (Cyclophilin-B).Specific primer/probe sequences are available upon request. For RNA-Seq,libraries were prepared using the Illumina TruSeq Stranded Total RNASample Preparation kit according to the manufacturer's protocol.Singled-end sequencing (50 bp) was performed on pooled libraries ingroups of 3 using an Illumina HiSeq 2500.

RNA-Seq and ChIP-Seq Analyses.

Sequencing reads generated from the Illumina platform were assessed forquality using FastQC. The reads were then trimmed for adapter sequencesusing TrimGalore. For RNA-Seq, reads that passed quality control werethen aligned to rn6 using TopHat (21). The TopHat results were thenanalyzed for differential expression using Cufflinks to generate thefragments per kilobase of exon per million fragments mapped (FPKM) foreach gene (22). Differential genes were identified using a significancecutoff of q<0.005 and fold change >1.5. These genes were then subjectedto further analysis. All original RNA-Seq data were deposited in theNCBI's GEO (GSE98575). For ChIP-Seq, the reads that passed qualitycontrol were aligned to mouse genome release mm9 using Burrows-WheelerAlignment (BWA) (23). Peaks were called using MACS 1.4.2 (24). Thedefault P value for peak detection was used (1×10−5). Further annotationand analysis of the called peaks were performed using HOMER, usingdefault settings. For each immunoprecipitated sample, a matchingchromatin input sample was used.

Hierarchical Clustering

The hierarchical relationship for the samples was determined usingdifferentially expressed genes between all pairwise comparison groups.The samples were clustered using Hierarchical-

Clustering v6 (Broad Institute) by pairwise average-linkage according tothe distance measure using the Pearson correlation coefficient. Heatmapsand dendrograms were generated from the output of theHierarchicalClustering using Java TreeView.

GSEA

Enriched pathways were determined using GSEA v17.6 using GenePattern andthe Molecular Signature Database (MSigDB; Broad Institute). The FPKMvalues from significantly differentially expressed genes (q<0.005 andfold change >1.5) as determined from the Cufflinks software werenormalized for each gene across all samples, and the Z score was used asinput for pathway analysis. The gene set used for comparison was theKEGG database, and enrichment significance was determined using 1000genomes project. Pathways were considered enriched using a significancecutoff FWER <0.25.

Gene Ontology Analysis

KEGG analysis were performed using DAVID bioinformatics suite.

Mitochondrial Respiration Sstudies

The assay was performed using Seahorse (Agilent) and following themanufacturer's published protocol. Isolated mitochondria (0.5 μg;measured by BCA assay) were loaded per assay.

Mitochondrial Genome Quantification

Total DNA was extracted from the hearts or NRVMs using the QlAamp DNAMini Kit (Qiagen, 51304). Mitochondrial DNA content was assessed by qPCRusing primers specific for multiple mitochondrial-encoded genes(mt-Cox1, mt-Nd2, mt-Nd1, and mt-Nd5) and normalized to nuclear DNAcontent (a specific locus on mouse chromosome 6) using the ΔΔCt method.

Transmission Electron Microscopy

Small pieces of tissue from the LV free wall were fixed by sequentialimmersion in triple aldeh yde-DMSO, ferrocyanide-reduced osmiumtetroxide, and acidified uranyl acetate; dehydrated in ascendingconcentrations of ethanol; passed through propylene oxide; and embeddedin Poly/Bed resin (Polysciences Inc., 21844-1). Thin sections weresequentially stained with acidified uranyl acetate, followed by amodification of Sato's triple lead stain, and examined with a JEOL1200EX electron microscope.

Results

To start deciphering its function in the heart, we analyzed ourpreviously reported REV-ERBα cistrome in the heart. Similar to previousreports in the liver and macrophages, the top enriched motifs areREV-ERBα's own DNA cognate sites (ROR and Reverb, DR2), followed bytissue-specific factor sites, MEF2a and MEF2c. MEF2a and MEF2c are knowndrivers of the cardiac hypertrophy gene program, and previous studiesshow that ectopic expression of MEF2a or MEF2c in the heart leads todilated cardiomyopathy and HF. They are also the top enriched enhancermotifs that undergo chromatin state switching in a murine cardiacpressure overload model. The colocalization of REV-ERBα to MEF2a andMEF2c suggests that it may specifically repress the aberrantly activatedgene program during cardiac hypertrophy and HF driven by MEF2a andMEF2c. SR9009 is a validated synthetic REV-ERB agonist, whichfacilitates REV-ERBα to recruit its corepressor NCoR and repressdownstream targets. We first tested the effect of SR9009 directly inprimary cardiomyocytes in vitro. In neonatal rat ventricular myocytes(NRVMs), SR9009 effectively blocked the phenylephrine-induced(PE-induced) cellular hypertrophy and expression of cellular stressmarkers (FIG. 1 , A-C). Further, another independently developed,structurally distinct REV-ERB agonist, GSK4112, also showed a similarresult (FIG. 2D). Thus, activation of REV-ERB leads to blockade ofcardiomyocyte hypertrophy and cellular stress in a cell-autonomousfashion.

We subsequently focused our study using SR9009 due to its higherefficacy and favorable in vivo pharmacodynamics. To elucidate themolecular mechanisms underlying REV-ERBα effects, we performed RNA-Seqon NRVM at baseline and after PE stimulation (4 hours [hr] and 48 hr) inthe presence or absence of SR9009 (SR or Veh were given 24 hr prior toPE). All original RNA-Seq data were deposited in the NCBI's GeneExpression Omnibus (GEO GSE98575). All the genes with pairwisedifferential expression between SR9009 and vehicle-treated (Veh-treated)groups at each time point were analyzed using Gene Set EnrichmentAnalysis (GSEA), and the genes in the altered pathways (defined byfamily-wise error rate [FWER]<0.25) were further analyzed usingunsupervised hierarchical clustering. Veh-PE-treated groups showed ashift in gene expression patterns at 4 hr that continued to deviate fromthe baseline with time. In contrast, the SR-treated groups displayed anexpression pattern more similar to the baseline groups, despite thepersistent exposure to PE for 48 hr. We then compared the number ofdifferentially expressed genes in each group (vs. Veh-baseline anddefined by changes >1.5-fold and q<0.005).

We found that, while the Veh-treated groups have about equal number ofgenes being up- or downregulated, the SR-treated groups have twice asmany genes being downregulated than upregulated, consistent with itsrole as a transcriptional repressor. Further, as the total number ofdifferential genes continue to increase from 4 hours of vehicletreatment (Veh-4h) to Veh-48h, indicative of the continuation of thehypertrophy process, the SR-48h had significantly fewer differentiallyexpressed genes compared with the early time point SR-4h, suggesting thehypertrophy process was blocked at the transcriptional level.Representative genes with REV-ERBα and MEF2a cooccupancy andtranscription repression by SR9009 treatment were shown. Using GSEA, weanalyzed the differentially expressed genes between SR9009 and Veh ateach time point. Baseline and 4-hr time points were combined, as theyshowed the same top enriched pathway (hypertrophic cardiomyopathy) whenanalyzed individually. Veh-treated cells showed an enrichment forcardiomyopathy and contractile pathways at baseline and 4 hr. Theactivation of hypertrophic pathways at baseline in the Veh-treated groupsuggests that, under the current culture condition (without PE), thereis a low level of spontaneous hypertrophy that is prevented by SR9009treatment.

Forty-eight hr after PE exposure, the Veh-treated group showed anenrichment for remodeling and inflammation pathways most consistent withthe advanced hypertrophic stage, while the SR9009-treated group showedan enrichment of cellular metabolism pathways known to be downregulatedin the failing heart. These results suggest that the REV-ERB agonistexerts its effect mainly through gene repression and that the differencebetween the SR9009 and Veh groups lies in the genes and pathways thatare up- or downregulated in the Veh groups and less changed in theSR9009 groups, as Nppa and Nppb show in FIG. 1D.

As a large number of genes involved in the fatty acid oxidation pathwaymaintained their expression with SR9009 treatment, we investigated therole of PDK4, an important modulator of the pathway. Interestingly, weobserved a cooccupation of MEF2A and REV-ERBα on the promoter and anenhancer element at the Pdk4 locus. In addition, PE represses theexpression of Pdk4, which is ameliorated by SR9009 treatment, as seen inour RNA-Seq result. PDK4 is likely one of themain targets of REV-ERBαduring cardiac metabolic remodeling.

Given the strong effect of SR9009 in blocking the cellular remodeling inresponse to neurohormonal stress, we next tested the role of REV-ERBαactivation in vivo using the classical pressure overload model. Westarted daily i.p. injection of SR9009 or Veh 1 day after transaorticconstriction (TAC, 27 gauge) surgery and monitored cardiac hypertrophyand function by echocardiogram for 6 weeks. The SR9009 dose (10mg/kg/day) was previously established and displayed no apparenttoxicity. We found that, while mice treated with Veh demonstrated agradual reduction in cardiac function (ejection fraction [EF]=52.8% at 4weeks and EF=46.9% at 6 weeks), SR9009-treated animals maintained normalcardiac function (EF=61.4% at 4 weeks and EF=60.6% at 6 weeks, P=0.03)(FIG. 2 , A-D). The SR9009-treated group also showed significantly lesscardiac hypertrophy measured by echocardiogram. The corrected leftventricle mass (LVm) showed a continued increase in the Veh-treatedgroup (LVm=129.6 mg at 4 weeks and LVm =139.2 mg at 6 weeks), which isattenuated in the SR9009-treated group (LVm=111.4 mg at 4 weeks [P=0.01]and LVm=114.8 mg at 6 weeks [P=0.02]). The left ventricle posterior wallthickness (LVPW; diastole [d]) and intraventricular septum thickness(IVS; d) showed similar trends (FIG. 3A). These findings were laterconfirmed by autopsy performed 6 weeks after TAC by heart weight andmuscle fiber size analyses using wheat germ agglutinin staining oncryopreserved sections (FIG. 3 , B-E).

Furthermore, we observed drastically reduced intracardiac fibrosis inthe SR9009-treated group. At the end of 6 weeks after TAC, theVeh-treated group showed an intramuscular fibrosis area of 10.3%, whilethe SR9009-treated groups showed no significant fibrosis, with anintramuscular fibrosis area of 1.1%, P=0.003 (FIG. 3 , F and G). Celldeath quantified by TUNEL assay also indicated that there is more celldeath in the Veh-treated group (27.8 tunnel positive cells percross-section of the whole heart) than the SR9009-treated group (13.4positives per cross-section of the whole heart, P=0.002) (FIG. 3 , H andI).

SR9009 is a drug given systemically and may potentially affect othersystems, such as the vascular tone, although pressure overload itself isconsidered a relatively specific stress directly on the heart. Toexclude the possibility that the improved cardiac function and reducedhypertrophy in vivo is associated with improvement in hemodynamics, wemeasured blood pressure using tail cuff and found there is no differencein mice receiving 4 weeks of Veh (mean arterial pressure [MAP]=106 mmHg)or SR9009 (MAP=115 mmHg, P=0.24). Since there is a substantiallyimproved fibrosis in the SR9009-treated heart, we also investigated ifthe in vivo protection is directly on cardiomyocytes or through cardiacfibroblasts (CFs).

We found there is no difference in the TGFβ1-induced inflammatoryresponse of cultured CFs pretreated with Veh or SR9009. Further, we seeno difference in capillary density after 6 weeks of TAC between Veh- andSR9009-treated groups, suggesting there is no secondary benefits throughimproved angiogenesis and microcirculation. Thus, REV-ERB agonism has adirect protection on the cardiomyocytes, consistent with our NRVMresults in culture. Using RNA-Seq, we found that in NRVM SR9009-treatedcells showed an enrichment in metabolic pathways that are known to bedownregulated in HF, in particular fatty acid metabolism, which accountsfor 70% of the energy source in the normal heart. Also, SR9009 haspreviously been reported to increase mitochondrial number, function, andmitophagy in skeletal muscle. We then investigated if there is a directinduction of mitochondria metabolism by SR9009. However, even after 10weeks after TAC in the mouse hearts, there is no difference inmitochondria number estimated by mtDNA to nuclear DNA ratio, nodifference in morphology by electron microscopy, and no difference inoxidative phosphorylation (OXPHOS) measured by oxygen consumption rate(Supplemental FIG. 1 , E-G). These observations indicate that animproved energy metabolism is not the direct cause of REV-ERB-inducedcardiac protection; rather, they support our cistrome and transcriptomicdata that a pathological gene program is repressed in thecardiomyocytes.

We next sought to determine if REV-ERBα activation impacts the failingheart. Most interventions using animal models have focused on theoutcome of disease prevention. However, patients often present withexisting and sometimes late-stage HF; a potential therapy in humanpatients requires efficacy in treating established or even late-stagedisease. To establish an HF model, we performed a 28-gauge TAC (moresevere than the commonly used 27-gauge model) on a cohort of 18 mice(FIG. 4 ). At 2 weeks after TAC, we observed an average EF of 44% ±1.3%in all the surviving mice (n=10). We randomized them into 2 groups basedon EF and treated 1 group with Veh (Group I) and another with SR9009(Group II). At 6 weeks after TAC, Group II maintained cardiac function,with an EF of 43.9% ±5%, while Group I dropped to 29.1% ±1.8% (P =0.01).

We then performed a cross-over, in which Group I received SR9009 andGroup II received Veh, and we observed animals for an additional 5weeks. Group I maintained an EF of 29.8% ±3.7%, while Group II droppedto 17.5% ±2.3% (P=0.01) (FIG. 3 , F and G). This result suggests thatthe REV-ERBα pathway can halt moderate and even severe HF. Finally, weinvestigated if a similar modulatory role of REV-ERBα exists in humancardiomyocytes.

We induced cellular hypertrophic response in human induced pluripotentstem cell-differentiated cardiomyocytes with endothelin-1 (ET-1) andmonitored NPPB (BNP) expression with SR9009 or Veh pretreatment. Not toosurprisingly, we observed a protective effect of SR9009, just as in therodent cardiomyocytes (FIG. 5 ).

This example shows that REV-ERBα is a key regulator of cardiacpathological remodeling and ameliorates HF, both in a preventative andtherapeutic fashion, in rodent models. Mechanistically, we demonstratedthat REV-ERBα colocalizes with driver TFs, such as MEF2a and MEF2c, andrepresses aberrant gene expression during cardiac pathological stress.

Multiple TFs and chromatin remodeling factors have been shown toparticipate in the cardiac hypertrophy gene program. Using heart tissueChIP-Seq, we have found that REV-ERBα can colocalize with driver TFs andcoordinate transcription repression at thousands of loci in the genomemediated by multiple TFs, which can prevent pathogenic switch of geneprogram.

Several compounds have been generated to manipulate REV-ERB function. Wechose to use SR9009 for its high efficacy and feasibility for in vivostudies. The specificity of SR9009 to REV-ERB has been shown to bind all48 human nuclear receptors. Previously, SR9009 has been used in otherstudies and has demonstrated effects in shortening sleep, reducinganxiety, increasing skeletal muscle performance and endurance exercise,increasing oxygen consumption, and protecting against obesity induced byhigh fat diet. Due to its effect on mitochondria number, function, andmitophagy in skeletal muscle, as well as our GSEA results indicating anenriched expression in fatty acid metabolism, we tested the possibilityof mitochondrial-driven cardiac protection. Our results indicate thatthere is no significant benefit directly associated with increasedmitochondrial number or function. This difference may be due to tissuespecificity or the particular type of injury being used. A more directinjury on the mitochondria, such as ischemia reperfusion, may inducemore observable changes in the mitochondria.

Although pressure overload is a relatively heart-specific injury model,other systems and cell types may play a significant role in the outcome.We show that there is no difference in blood pressure, mitochondrianumber/morphology/oxidation and phosphorylation, capillary density, orCFs, suggesting that the SR9009-treated hearts had preserved cardiacfunction due to less cardiomyocyte injury instead of any secondarybenefit from improved hemodynamics, metabolism, microcirculation, orless-reactive CFs. We conclude that REV-ERB activation has a directprotective effect in cardiomyocytes in a cell-autonomous fashion. Werealize that fibrosis in vivo requires interaction between thecardiomyocytes and CFs, as well as other secretory factors.

We have shown that, in mice, the therapy is effective even in advanceddisease at least to prevent disease progression—despite of thepersisting insult. Its efficacy in advanced disease is particularlyattractive to provide medical management to end-stage HF patients inorder to avoid or delay heart transplant or mechanical assisting device.

While this invention has been shown and described with references tovarious embodiments thereof, it will be understood by those skilled inthe art that changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims. All patents, publications and references cited in the foregoingspecification are herein incorporated by reference in their entirety.

What is claimed is:
 1. A method of treating cardiac hypertrophy in asubject in need thereof, the method comprising: administering to thesubject a therapeutically effective amount of a REV-ERBα agonist.
 2. Themethod of claim 1, the REV-ERBα agonist selected from the groupconsisting of 1,1-DimethylethylN-[(4-chlorophenyl)methyl]-N-[(5-nitro-2-thienyl)methyl])glycinate(SR6452);N-Benzyl-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;N-Benzyl-N-(3,4-dichlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N,N-dimethylacetamide,SR9009, SR9011 and pharmaceutically acceptable salts thereof.
 3. Themethod of claim 2, the REV-ERBα agonist selected from the groupconsisting of SR9009 and SR6452.
 4. The method of claim 1, wherein thetherapeutically effective amount is the amount required to inhibitcardiomyocyte hypertrophic growth in the subject.
 5. The method of claim1, wherein the therapeutically effective amount is the amount requiredto reduce cardiac fibrosis and/or cardiac cell death in the subject. 6.The method of claim 1, wherein the REV-ERBα agonist is administered tothe subject by at least one of oral and/or parenteral delivery.
 7. Amethod of treating heart failure in a subject in need thereof, themethod comprising: administering to the subject a therapeuticallyeffective amount of a REV-ERBα agonist.
 8. The method of claim 7, theREV-ERBα agonist selected from the group consisting of 1,1-DimethylethylN-[(4-chlorophenyl)methyl]-N-[(5-nitro-2-thienyl)methyl])glycinate(SR6452);N-Benzyl-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;N-Benzyl-N-(3,4-dichlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N,N-dimethylacetamide,SR9009, SR9011 and pharmaceutically acceptable salts thereof.
 9. Themethod of claim 8, the REV-ERBα agonist selected from the groupconsisting of SR9009 and SR6452.
 10. The method of claim 7, wherein thetherapeutically effective amount is the amount required to inhibitcardiomyocyte hypertrophic growth in the subject.
 11. The method ofclaim 7, wherein the therapeutically effective amount is the amountrequired to reduce cardiac fibrosis and/or cardiac cell death in thesubject.
 12. The method of claim 7, wherein the REV-ERBα agonist isadministered to the subject by at least one of oral and/or parenteraldelivery.
 13. A method of inhibiting cardiac fibrosis in a subject inneed thereof, the method comprising: administering to the subject atherapeutically effective amount of a REV-ERBα agonist.
 14. The methodof claim 13, the REV-ERBα agonist selected from the group consisting of1,1-DimethylethylN-[(4-chlorophenyl)methyl]-N-[(5-nitro-2-thienyl)methyl])glycinate(SR6452);N-Benzyl-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;N-Benzyl-N-(3,4-dichlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine;2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)-N,N-dimethylacetamide,SR9009, SR9011 and pharmaceutically acceptable salts thereof.
 15. Themethod of claim 14, the REV-ERBα agonist selected from the groupconsisting of SR9009 and SR6452.
 16. The method of claim 13, wherein thetherapeutically effective amount is the amount required to inhibitcardiomyocyte hypertrophic growth in the subject.
 17. The method ofclaim 13, wherein the therapeutically effective amount is the amountrequired to reduce cardiac cell death in the subject.
 18. The method ofclaim 13, wherein the REV-ERBα agonist is administered to the subject byat least one of oral and/or parenteral delivery.